Project Summary: The gut microbiome comprises hundreds of bacterial taxa that modulate intestinal health, immunity, and metabolism. Intriguingly, emerging studies have reported that supplementation of beneficial bacteria, known as ?probiotics?, can elicit positive salutary effects on intestinal health and disease. Our research group has been focused on discovering the underlying molecular mechanisms whereby probiotics elicit their positive effects on the intestinal mucosa. In these studies, we have focused on the most widely studied probiotic strain, Lactobacillus rhamnosus GG (LGG). As well as preserving the well-being of a healthy intestine, studies have reported that the microbiome, and specifically LGG, can influence tumor burden in models of colon carcinogenesis in mice; however, the immunological mechanisms involved are still in need of greater investigation. In addition, the microbiome has been shown to effect patient?s response rates to immunotherapy in the treatment of cancer and probiotics such as LGG have been shown to modulate the microbiome. Furthermore, LGG and the microbiome at large has been shown to elicit robust effects on the mucosal immune response. Based on these observations, our rationale is that LGG exerts a beneficial response in colon cancer through mechanisms involving the modulation of cytotoxic immune pathways that respond to tumors and that this would synergize with immunotherapy. In support of this, our preliminary data demonstrate that feeding LGG can reduce tumor burden in the DSS-AOM mouse model of colitis-associated carcinoma when administered after the emergence of tumors. In addition, analysis of CD8 T-cells in healthy mice show that LGG can induce the expression of cytotoxic, effector CD8 T-cells via a MyD88-dependent mechanism. Therefore, we hypothesized that LGG can induce cytotoxic immune infiltrate into colonic tumors and that this induction will synergize with immunotherapy. We will test our hypothesis by the following aims. 1. To identify the molecular mechanism and magnitude whereby LGG induces an enhanced effector CD8 T-cell response in colon cancer. We will establish if LGG can induce similar CD8 T-cell responses in colonic tumor-bearing mice, as detected in healthy mice. Because LGG upregulates TLR2 expression, and activates T-cells in a MyD88-depndent manner, we will also test if LGG induction of CD8 T-cells is TLR2-dependent. Finally, we will establish if this induction of CD8 T-cells is necessary for LGG?s protective role in colon cancer animal models. 2. To identify the co-therapeutic efficacy of LGG on inflammatory colon tumors. We will establish if LGG-mediated reduction of tumor burden and expansion of CD8 T-cells can synergize with anti-PD1 immunotherapy. Additionally, as anti-PD1 responses have been linked to changes in the microbiome, we will analyze the microbiome of tumor-bearing mice after LGG administration, and test if this microbiome is sufficient at eliciting the same tumor-reductive effects when transferred into germ-free mice. Overall, understanding these mechanisms would validate the use of LGG as a co-therapy with anti-PD1 and substantiate the microbiome?s effect on mucosal immune responses and immunotherapy response rates.